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Rapid Cryogenic Fixation of Biological Specimens for Electron Microscopy

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RAPID CRYOGENIC FIXATION OF BIOLOGICAL SPECIMENS FOR ELECTRON MICROSCOPY KEITH PATRICKRYAN A thesis submitted in partial fulfilment of the requirements of the Council for National Academic Awards for the degree of Doctor of Philosophy September 1991 Polytechnic South West in collaboration with the Marine Biological Association of the United Kingdom and Plymouth Marine Laboratory ----- . \ ~ ,, '' - .... .._~ .. ·=·~-·-'-·-'" --······ --~....... ~=.sn.-.......... .r.=-..-> POL VTECHNIC SOUTH WEST liBRARY SERVICES Item C!O 00 7 9 4 9 3-0 No. ', )'; I Class 1 !) -, C!f -RYA !No. rr ... · ,Contl No. :x70ZS\0253 ·. ' COPYRIGHT This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the authors prior written consent. 2 CONTENTS Page List of Figures 8 List of Tables 10 Abstract 11 Acknowledgements 12 1 Introduction 13 2 Literature Review 23 2.1 Background to specimen preservation for microscopy 23 2.2 Problems of chemical processing for electron rhlcroscopy 23 2.3 Introduction of cryotechniques into microscopy methods 24 2.4 The potential of cryofixation 25 2.5 The problems of cryofixation 25 2.6 Water, cooling and crystal nucleation 26 2. 7 Cell water 29 2.8 Crystallisation and latent heat release 29 2.9 Phase separation and eutectic temperature 30 2.10 Types of ice 31 2.11 Phase transitions 32 2.12 Ice crystal growth in frozen specimens after freezing 34 2.13 Cryoprotection against ice crystal damage 37 2.14 Modelling the cooling process 38 2.15 Cooling methods 45 2.16 Coolants (liquid) 46 2.17 Coolants (solid) 46 2.18 Plunge cooling methods 48 2.19 Jet cooling methods 51 2.20 Cryoblock methods 54 2.21 Rapid cooling experiments 59 3 2.22 Specimen rewarming during handling after freezing 74 2.23 Conclusions 74 3. Materials and Methods 78 3.1 Coolants 78 3.2 Safety 78 3.3 Liquefaction of gases 80 3.4 Temperature control of coolants 82 3.5 Thermocouples 83 3.6 Thermocouple construction 84 3.7 Thermocouple sensitivity 89 3.8 Plunge velocity sensor 89 3.9 Recording and display electronics 91 3.10 Calculation of cooling rates 93 3.11 Calculation of plunge velocity 94 3.12 Hydrated gelatin-thermocouple specimens 94 3.13 Metal-sandwiched gelatin-thermocouple specimens 99 3.14 Metal-sandwiched hydrated gel & blood specimens 101 3.15 Freeze-substitution 101 3.16 Transmission Electron Microscopy (TEM) 102 3.17 Arrowworm (Sagitta) specimens 102 3.18 Low thermal mass supports 103 3.19 Specimens for monitoring ice crystal growth 103 3.20 Cryoglues for cryomounting 105 3.21 Carrot (Daucus) specimens 105 3.22 Scanning Electron Microscopy (SEM) 105 3.23 Cryo-stage 106 3.24 Energy Dispersive X-Ray Microanalysis 106 3.25 Freeze-drying 107 3.26 Statistics and n =2 to n =6 108 4 4 Cooling in metal specimens (bare thermocouples) 110 4.1 Summary 110 4.2 Introduction 110 4.3 Experimental Design 111 4.4 Results 115 4.5 Discussion 115 4.6 Conclusions 121 5 Cooling in exposed, hydrated specimens 122 5.1 Summary 122 5.2 Introduction 122 5.3 Experimental Design 124 5.4 Results 127 5.5 Discussion 132 5.6 Conclusions 136 6 Cooling in metal-sandwiched, hydrated specimens 137 6.1 Summary 137 6.2 Introduction 137 6.3 Experimental Design 138 6.4 Results 141 6.5 Discussion 142 6.6 Conclusions 153 7 Crystal growth in metal-sandwiched, hydrated specimens 155 7.1 Summary 155 7.2 Introduction 155 7.3 Experimental Design 157 7.4 Results 158 7.5 Discussion 158 7.6 Conclusions 170 5 8 Cryofixation and analysis of Chaetognath specimens 172 8.1 Summary 172 8.2 Introduction 172 8.3 Experimental Design 173 8.4 Results 177 8.5 Discussion 177 8.6 Conclusions 186 9 Cryomounting of frozen specimens for cryoSEM 187 9.1 Summary 187 9.2 Introduction 187 9.3 Experimental Design 189 9.4 Results 193 9.5 Discussion 193 9.6 Conclusions 198 10 The effect of exposure to subzero processing temperatures 200 10.1 Summary 200 10.2 Introduction 200 10.3 Experimental Design 201 10.4 Results 203 10.5 Discussion 206 10.6 Conclusions 210 11 The rate of cryosubstitution 211 11.1 Summary 211 11.2 Introduction 211 11.3 Experimental Design 213 11.4 Results 214 11.5 Discussion 218 11.6 Conclusions 219 12 General conclusions 221 6 Bibliography 229 Appendices: 1 Collaborators 254 2 Activities undertaken in connection with this research 256 3 List of publications associated with this research 260 4 Copies of published papers: 4.1 A simple plunge-cooling device for preparing biological specimens for cryo-techniques 261 4.2 The relative efficiency of cryogens used for plunge-cooling biological specimens 266 4.3 Cooling rate and ice crystal measurement in biological specimens plunged into liquid ethane, propane and Freon 22. 27 4 4.4 On the differences between different "indicator" species of Chaetognath, Sagitta setosa and S. elegans 288 7 List ofFigures Page 1 Liquefaction of gases 81 2 Thermocouple construction 85 3 Scanning electron micrograph of a fine thermocouple 86 4 Photograph of the 300 J.lm diameter thermocouples 88 5 Plunge velocity sensor 90 6 Recording and display electronics 92 7 Calculation of cooling rate and plunge velocity 95 8 Thermocouple response in liquid nitrogen 96 9 Constructing hydrated gel specimens 98 10 Metal-sandwiched hydrated gel/thermocouple specimen 100 11 Low thermal mass specimen supports 104 12 Benchtop plunge-freezing device - I (diagram) 112 13 Benchtop plunge-freezing device - II (photograph) 114 14 Typical record from the benchtop plunger 116 15 Determinants of cooling rates 123 16 Deep plunging device 125 17 Typical record from the deep plunger 128 18 Cooling rates in exposed specimens 130 19 Cooling distances of exposed specimens 131 20 Back-scattered electron image of the sandwiched specimen 140 21 Cooling record from a metal-sandwiched gel specimen 143 22 Cooling rates in a metal-sandwiched gel specimen 144 23 Cooling curves from a resin specimen and a gel specimen 148 24 Rewarming of a specimen by latent heat during freezing 149 25 Cooling distances for the metal-sandwiched gel specimen 151 26 Ice damage in a freeze-substituted gel specimen 159 27 Transects of ice profile size across gel specimens 160 8 28 Ice damage in freeze-substituted blood cells - I 162 29 Ice damage in freeze-substituted blood cells - 11 163 30 Ice damage in freeze-substituted blood cells - Ill 164 31 Ice damage in freeze-substituted blood cells - IV 165 32 Transects ofice profile size across blood samples 166 33 CryoSEM image of Sagitta 178 34 X-ray spectra from frozen Sagitta specimens 179 35 ZAF PB computer printout of a typical X-ray analysis 180 36 Electron micrographs of surface cryofixation 182 37 Electron micrographs of centre-line-cryofixation 183 38 The centre-line-cryofixation principle 186 39 Cryomounting chamber (diagram) 190 40 Cryomounting system (photograph) 192 41 CryoSEM image of a cryomounted Sagitta 194 42 CryoSEM image of a non-cryomounted Sagitta 195 43 Anaglyph of a carrot cell showing the ice cavity 197 44 Frozen cells stored at high subzero temperatures - I 204 45 Frozen cells stored at high subzero temperatures - ll 205 46 Cryofixed spleen stored at 213 K (-60°C) for 48 hours 207 47 Partial pressures recorded during freeze-drying 215 48 Images of uranium penetration during freeze-substitution 216 49 The penetration of uranium during freeze-substitution 217 9 List of Tables Page 1 Coolants introduced for rapid freezing in microscopy 47 2 Cooling rates from a bare thermocouple 117 3 Heat transfer coefficients 120 4 Cooling rates from an exposed, hydrated specimen 129 5 Measured and theoretical plunge depths 145 6 Cooling rates from metal-sandwiched specimens 146 7 Measured and predicted ice crystal sizes 161 8 X-ray microanalysis results 181 10 RAPID CRYOGENIC FIXATION OF BIOLOGICAL SPECIMENS FOR ELECTRON MICROSCOPY KEITH PATRICK RYAN Abstract This thesis describes investigations into cryofixation by the plunge­ cooling technique, at ambient pressure. The objective was to characterise coolants which are commonly used for cryofixation, so that the structure and chemistry of biological specimens may be preserved in a more life-like state. The work began with the design of a suitable cooling device. This was developed further into a large test-bed apparatus which was used in both biological and methodological experiments. The large cooling apparatus demonstrated for the first time that ethane was a superior coolant under forced convection, compared to propane or Freon 22, for bare thermocouples, for exposed hydrated specimens and for metal-sandwiched hydrated specimens. Ice crystal formation was monitored in sandwiched specimens and found to correspond closely to modelling predictions. A biological application was the X-ray microanalysis of body fluids in "indicator" species of Chaetognaths, where results obtained from cryo­ scanning electron microscopy revealed ecophysiological differences. The use of low thermal mass supports demonstrated that good freezing can occur in the centre of specimens. A new cryomounting method was developed to load well-frozen specimens into the microscope. The effect of post-freeze processing temperature was investigated by monitoring ice crystals in red blood cells. Exposure to 213 K (-60°C) over a 48 hour period did not induce crystal growth and exposure to 233 K (-40°C) for 8 days showed minimal ice crystal damage. The progress of cryosubstitution was monitored over 48 h at 193 K (-80°C), this showed that uranium ingressed to a depth of 320 1J..D1 which could be doubled when shrinkage was allowed for.
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